US 7129037 B2
This invention relates to methods and compositions useful for delivering antigens to dendritic cells which are then useful for inducing antigen-specific cytotoxic T lymphocytes and T helper cells. This invention also provides assays for evaluating the activity of cytotoxic T lymphocytes. According to the invention, antigens are targeted to dendritic cells by apoptotic cells which may also be modified to express non-native antigens for presentation to the dendritic cells. The dendritic cells which are primed by the apoptotic cells are capable of processing and presenting the processed antigen and inducing cytotoxic T lymphocyte activity or may also be used in vaccine therapies.
1. A method of assessing cytotoxic T lymphocyte activity comprising:
a) providing an antigen presenting dendritic cell prepared by contacting the dendritic cell with an apoptotic cell expressing an antigen or an apoptotic cell fragment, bleb, or body containing an antigen, wherein the apoptotic cell is shown to be apoptotic by a procedure selected from the group consisting of Annexin V staining, propidium iodide staining, DNA laddering, and staining with dUTP and terminal transferase (TUNEL staining), wherein said contacting is for a time sufficient to allow said antigen to be internalized by the dendritic cell, and wherein said apoptotic cell expressing or containing said antigen has been induced in vitro to become apoptotic prior to exposure to the dendritic cell;
b) exposing the antigen presenting dendritic cell of step a) to a population of T lymphocytes to be assayed for their ability to exhibit killer cell activity; and
c) assaying the cytotoxic activity of the T lymphocytes exposed to said antigen presenting dendritic cell.
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This application is a continuation of U.S. application Ser. No. 09/251,896, filed Feb. 19, 1999, now U.S. Pat. No. 6,602,709, which claims priority to U.S. provisional applications Ser. No. 60/075,356, filed Feb. 20, 1998; Ser. No. 60/077,095, filed Mar. 6, 1998; and Ser. No. 60/101,749, filed Sep. 24, 1998, all of which are incorporated herein by reference in their entireties.
This invention was made with United States Government support under National Institutes of Health grant AI-39516, AI-13013 and AI 39672. The United States Government has certain rights in this invention.
This invention relates to targeted antigen presentation in the immune system. In particular, this invention relates to the use of apoptotic cells to deliver antigens to dendritic cells for induction or tolerization of antigen-specific T cell responses. The apoptotic cell-mediated antigen delivery system described herein has a wide range of preventive, diagnostic and therapeutic applications.
CD8+ cytotoxic T lymphocytes (CTLs) play a critical role in immune defense against infectious agents, tumors and transplants. Class I-restricted CD8+ T cells have been implicated in the recognition and destruction of such clinically important targets as HIV-1 (1–3), Influenza A (4), malaria (5), cytomegalovirus infected cells (6), Epstein-Barr virus (7), and human melanoma cells (8–9). Therefore, establishing methods for inducing and expanding populations of antigen-specific CD8+ CTLs remains an important objective in the development of therapeutic treatments against infectious disease and cancer.
CD8+ CTLs are activated by antigens which have been processed and presented on major histocompatibility (MHC) class I molecules on the surface of specialized antigen presenting cells. A number of antigen presenting cells have been identified which activate T cells including macrophages/monocytes, B cells and bone marrow derived dendritic cells. Of these, dendritic cells are recognized as playing a pivotal role in the initiation of CD8+ CTL responses (10).
An important feature of dendritic cells is their ability to efficiently process and present antigens on MHC class I and/or class II molecules. Depending on the antigen processing pathway, dendritic cells are capable of activating distinct populations of CTLs. In the case of influenza virus, for example, it is known that the class I pathway for inducing CD8+ CTLs requires adequate delivery of infectious viral antigen into the cytoplasm, whereas the purely endocytic pathway delivers noninfectious virions for presentation only to CD4+ T helper cells (U.S. Ser. No. 08/282,966). Thus, although dendritic cells efficiently activate class I-restricted CTLs, access to the MHC class I pathway for induction of CD8+ T cells normally requires endogenous synthesis of antigen. Accordingly, it is important to identify antigen delivery systems which efficiently mediate access of exogenous antigen to the MHC class I-restricted antigen presentation pathway in order to generate antigen-specific CD8+ T cell responses.
Recently, a number of approaches have been reported for delivery of exogenous antigen to the MHC I processing pathway of dendritic cells. These methods include coupling antigens to potent adjuvants (11–15), osmotic lysis of pinosomes after pinocytic uptake of soluble antigen (16), or insertion of antigen in pH-sensitive liposomes (17). However, these prior approaches still pose a number of limitations on the development of effective therapeutic treatments. For example, Nair et al. report that dendritic cells do not efficiently internalize antigen-containing liposomes in vivo (18). Further, osmotic lysis of dendritic cell pinocytic vesicles may be difficult to perform in vivo, and may result in inefficient antigen delivery to the MHC class I processing compartment. Also, the use of powerful adjuvants may be undesirable for some clinical applications.
Pulsing dendritic cells directly with exogenous antigen using whole cells in viable or irradiated forms, membrane preparations, or antigens purified from natural sources or expressed as recombinant products has also been previously reported (WO 94/02156). These prior methods, however, do not recognize forms of cell death or the processing pathways antigens from dead or dying cells access in the dendritic cell system.
Rubartelli et al. report that dendritic cells, unlike macrophages, fail to take up opsonized particles or necrotic cells in vitro, but can efficiently engulf cells undergoing apoptotic programmed cell death (19). The mechanism of internalization of apoptotic cells by dendritic cells, however, is different than in macrophages, indicating that results in the macrophage system are not necessarily predictive of dendritic cell responses. In addition, Rubartelli et al. does not show presentation of engulfed material and therefore speculates as to the fate of such material.
This invention provides highly efficient methods for delivering exogenous antigens to dendritic cells and inducing antigen-specific T cell activation. In particular, the methods described in this invention are directed toward developing therapies for increasing patient immunity to chronic infections and tumors by 1) inducing tumor or infected cells to undergo apoptosis, 2) having the apoptotic tumor or infected cells gain access to phagocytic, maturing dendritic cells, and 3) exposing the apoptotic cell-primed dendritic cells expressing antigen of interest to T cells, in vivo or in vitro, for induction of antigen-specific T cell responses.
This invention further provides that the population of donor cells expressing said antigen can be induced to undergo apoptosis using a variety of methods including, but not limited to, viral infection, irradiation with ultraviolet light, gamma radiation, cytokine treatment, or depriving donor cells of nutrients in the cell culture medium. It is also contemplated by this invention that the dendritic cells can be exposed to a preparation of donor apoptotic cell fragments, blebs or bodies rather than whole apoptotic cells.
In another embodiment of this invention, the donor cells can be transfected, transduced or transformed to express foreign antigens prior to induction of apoptosis. A variety of such antigens may be expressed by the donor cells including, but not limited to, viral antigens, tumor antigens, toxins, microbial antigens, and autoimmune antigens.
Accordingly, this invention also provides a method of generating antigen-specific cytotoxic T lymphocytes comprising providing a population of apoptotic cells, or membrane containing fragments thereof, expressing said antigen, exposing dendritic cells to said apoptotic cells for a time sufficient to allow said antigen to be internalized and processed by the dendritic cells, and exposing T lymphocytes in vivo to said dendritic cells for a time sufficient to induce said lymphocytes to become antigen-specific T lymphocytes. This invention further contemplates induction of antigen-specific T lymphocytes in vitro.
In another embodiment of this invention apoptotic cells expressing an antigen to be presented by dendritic cells are administered to an individual in an amount and in a location so as to prime dendritic cells in vivo.
Methods of preventing and treating disease are also provided by this invention which comprises administering to an individual in need of treatment, a therapeutically effective amount of dendritic cells which have been primed by apoptotic cells.
In addition to the methods of this invention, this invention also provides dendritic cells which have been primed by apoptotic cells, and in particular, human dendritic cells which are prepared according to the methods of this invention. This invention further provides transformed apoptotic cells, or a preparation of transformed apoptotic cell fragments, and in particular, human apoptotic cells which are prepared according to the methods of this invention.
In another embodiment of this invention the apoptotic cell delivery system is reconstituted in vitro using liposomes comprising antigen and apoptotic cell proteins, factors or ligands which enhance uptake and processing of antigen in dendritic cells.
Based on the results disclosed herein, one may include in a liposome ligands for integrin receptors, including, but not limitd to the αvβ5 and αvβ3 integrin receptors for enhancing uptake and processing of antigens in dendritic cells. An example of such a ligand is lactadherin. Other examples of proteins which may enhance uptake of apoptotic particles include thrombospondin. Similarly, ligands which bind CD36 may also be used. In a preferred embodiment ligands for both integrin and CD36 would be used.
Heat shock proteins (HSP) may also be included to facilitate antigen uptake by dendritic cells. For example, HSP 70 family members, especially HSP 70, and HSP 90 members, especially HSP 84 and GP 96 may be used. Certain antigens obtained from infectious sources may already be bound to HSPs. Alternatively, antigens may be complexed to HSP ex vivo.
The identification of the αvβ5 integrin receptor as mediating antigen uptake by dendritic cells provides one with a means of modulating that antigen uptake by either increasing or decreasing the activity of the αvβ5 receptor. Accordingly, this invention provides means for enhancing antigen uptake and processing by dendritic cells in vitro or in vivo, by adding an αvβ5 ligand to the dendritic cells in conjunction with the antigen to be taken up and processed. Alternatively, one could attenuate the antigen uptake activity of the dendritic cells by blocking the αvβ5 receptor, or other receptors which are involved in medrating antigen uptake by the dendritic cells.
This invention also includes pharmaceutical compositions for charging dendritic cells comprising a ligand which activates antigen uptake, such as an αvβ5 ligand, with or without an antigen to be processed and presented by dendritic cells. As used in this invention such ligands may be considered to function as immunoadjuvants.
This invention also provides methods for modulating T cell responses in vivo or in vitro to induce immunity or tolerance by controling DC maturation.
In one embodiment, T cell immunity can be induced when immature DCs are caused to mature by the addition of appropriate maturation factors after the uptake of apoptotic particles. Examples of maturation factors that can be used in this invention include monocyte conditioned medium, TNF-α, IL-1β, IL6, PGE2, IFN-α, CD40 ligand, and necrotic cells.
In another embodiment, tolerance can be induced to self or foreign antigens by maintaining DCs in their immature state in the absence of a maturation signal.
It is a general object of this invention to provide a method of using apoptotic cells, or apoptotic cell fragments, to efficiently deliver specific antigens to dendritic cells which then process and present said processed antigens on their surface for stimulation or tolerization of cytotoxic T lymphocytes.
It is another object of this invention to generate antigen-specific cytotoxic T lymphocytes either in vivo or in vitro by using the dendritic cells generated by the methods described herein.
It is also an object of this invention to provide a method of prophylactic or therapeutic treatment for a variety of cancers, autoimmune diseases, and pathogens using the dendritic cells described herein.
[FIG 10B]: As in FIG 10 a, but DC-BALB/C blast co-cultures [top] or DCs only [bottom] were labeled with other mAbs to show that CD11c+ I-Ab+ I-Ad− DCs selectively acquired Y-Ae [arrows].
Freshly isolated, blood monocytesu were infected with live influenza A, PR/8 [Spafas Inc.], labeled with the PKH26-GL fluorescent cell linker compound [Sigma Biosciences], and incubated at 37° C. for 6–8 hours allowing apoptosis to occur. Macrophages, immature DCs and mature DCs were dyed with PKH67-GL and added to the culture wells containing the apoptotic monocytes at a ratio of 1:1. Cells were analyzed by FACScan® where double positive cells indicate uptake of the apoptotic cells by the various APCs [panels iii., vi., ix]. We used the various APCs alone to establish the proper settings [panels i., iv., vii.] Note, as the forward scatter of the APCs increased, the dying monocytes were excluded from the established region [panels ii., v., viii.]. After 2 hr., 80% of the macrophages 50% of the immature DCs, and less than 10% if the mature DCs had engulfed the apoptotic monocytes [A]. In an independent experiment, macrophages [squares], immature DCs [diamonds] and mature DCs [circles] were prepared and co-cultures with apoptotic monocytes were established as described above, and FACS® was performed at various time points. Percent phagocytosis was calculated based on the number of double positive cells [B].
Apoptotic monocytes and immature DCs were prepared as described. Immature DCs were pre-incubated at 4° C. [A], in the presence of varying concentrations of Cytochalasin D [B], or EDTA [C] for 30 minutes. Apoptotic monocytes were then added to the DC cultures at 4° C. [A] or 37° C. [B,C]. FACS® analysis was performed after 1–2 hrs. Data shown in
Influenza infected apoptotic monocytes were co-cultured with immature DCs for 1 hr after which the cells were adhered to a cover slip and fixed with acetone. Immunofluorescence was performed with anti-influenza nucleoprotein antibodies [NP] and Texas red conjugated goat anti-mouse IgG; and biotinylated anti-HLA-DR [DR] followed by FITC conjugated streptavidin. Large arrowhead indicates apoptotic cell outside the DC prior to engulfment. Small arrows indicate apoptotic material derived from the influenza infected monocytes within DR+ vesicles of the DC. These image were not generated on a confocal scope, so the structures of the DC underlying the apoptotic cell can be seen.
Various populations of HLA-A2.1+ antigen presenting cells [APCs] were co-cultured with HLA-A2.1− influenza infected monocytes. After 12 hrs, the APCs were loaded with 51Cr and used as targets for HLA-A2.1+ influenza-reactive CTL lines. Mature DCs were isolated by labeling with the DC-restricted marker CD83, followed by cell sorting on the FACSort® [Becton Dickinson]. Immature DCs were CD14− and sorted by FACSort® as a CD83− population. Mature macrophages were generated by culturing an adherent mononuclear cell fraction in a Teflon beaker for 9 days. Effector:Target Ratios=45:1 and 15:1 [A]. Controls included infected and uninfected mature DCs, immature DCs and macrophages. The HLA-A2.1− monocytes used as a source of apoptotic material were also tested as targets to demonstrate the absence of lysis when using a mis-matched target. Effector:Target Ratios=45:1 and 15:1. Results are representative of 3 experiments and the values shown represent the mean of triplicate wells [B].
Macrophages [A], immature DCs [B] and mature DCs [C] were prepared as previously described. Cells were incubated with anti-CD83, a DC maturation marker, either untreated or post-saponin treatment. The latter permeablized the cells allowing for intracellular staining. Cells were then labeled using a PE-conjugated GAM-Ig [Biosciences] A control isotype matched antibody was used.
Immature DCs [A] and mature DCs [B] were incubated with anti-αvβ3 [clone 23C6, Pharmingen], anti-CD36 [clone FA6, obtained from the Vth international workshop on leukocyte differentiation antigens], or anti-αvβ5 [clone P1F6, Chemicon], followed by PE-conjugated GAM-Ig [Biosciences]. All cells were analyzed by FACScan®. [C] RNA was purified from highly purified sorted cell populations of immature and mature DCs as previously described. RT-PCR was carried out and after 30 cycles of PCR the distinct bands for β3, β5 and CD36 could be seen in the immature DCs [lane 1]. In mature DCs only a faint band for CD36 and no band for β5 could be visualized [lane 2] indicating minimal mRNA. In contrast, a band was evident for β3 in the mature DCs. Note, the doublet for β3 is an artifact in this particular exposure and does not indicate two unique bands [lane 2]. As a positive control, extracts from Bowes melanoma cells were run, which are known to express β3 and CD36 (29) [lane 3]. As negative control, the Bowes melanoma cells were run in the absence of a reverse transcriptase [lane 4].
HeLa cells were labeled with PKH26-GL, followed by irradiation using a 60UVB lamp [Derma Control Inc.], calibrated to provide 240 mJ cm−2 in 2 minutes, sufficient for the induction of apoptosis. After 6–8 hours immature DCs dyed with PKH67-GL and pre-treated with 50 μg/ml of various monoclonal antibodies for 30 minutes were added to the wells containing apoptotic HeLa cells. 45–60 minutes later, cells were analyzed by FACS® for double positive cells. Phagocytic uptake is reported as a percentage of untreated cells. Maximal phagocytosis ranged from 44–52%. Results from three experiments were averaged and means plotted+SD. Similar results [data not shown] were obtained when apoptotic monocytes were used [A]. Immature DCs were incubated with red fluorescent latex beads at 37° C. [squares], 4° C. [diamonds], or at 37° C. in the presence of 50 μg/ml anti-αvβ5 [circles], anti-αv [triangles]. The results shown indicate percent phagocytosis over background [B].
This invention relates to methods of delivering antigens to dendritic cells for processing and presentation to T lymphocytes. In particular, this invention relates to the use of apoptotic cells for the packaging and delivery of exogenous antigen to the MHC class I presentation pathway of dendritic cells for stimulation or tolerization of class I-restricted CD8+ T cells. The apoptotic cell-activated dendritic cells and antigen-specific T cells produced according to the method of this invention may be used for various immunological interventions for the prevention and treatment of disease.
For the purpose of a more complete understanding of the invention, the following definitions are described herein:
The term “apoptosis” means non-necrotic cell death, which can occur under a variety of conditions including programmed cell death, exposure to ionizing and UV irradiation, activation of fas and other tumor necrosis factor receptor-related pathways, and drugs. Apoptosis is characterized by, inter alia, formation of “blebs” and vesicles at the plasma membrane, cell shrinkage, pyknosis, and increased endonuclease activity (20–21). Specific markers for apoptosis include, but are not limited to, annexin V staining, propidium iodide staining, DNA laddering, staining with dUTP and terminal transferase [TUNEL].
The term “apoptotic cell” means any cell expressing a native or foreign antigen undergoing apoptosis due to any condition, including those which ususally are associated with causing neurosis. Thus, an apoptotic cell is identified based on its characteristics described above rather than any method used leading to cell death. Similarly, the term “apoptotic cell fragments” means apoptotic cell material, bodies, blebs, vesicles, or particles other than whole apoptotic cells which contain antigen.
The term “necrosis” means a form of cell death resulting from irreversible trauma to cells typically caused by osmotic shock or exposure to chemical poison, and is characterized by marked swelling of the mitochondria and cytoplasm, followed by cell destruction and autolysis (22).
The term “donor cell” means the apoptotic cell that delivers antigen to dendritic cells for processing and presentation to T cells.
The phenomenon of “cross-priming” occurs when antigens from donor cells are acquired by the host APCs such as dendritic cells and are processed and presented on MHC molecules at the surface of the APC for activation of antigen-specific T cells.
The phenomenon of “cross-tolerance” occurs when antigens from donor cells are acquired by host dendritic cells and are presented under conditions that are non-inflammatory (lack of inflammation or other maturation stimuli) so as to cause antigen-specific unresponsiveness in T cells.
The term “antigen” means all, or parts thereof, of a protein or peptide capable of causing an immune response in a vertebrate preferrably a mammal. Such antigens are also reactive with antibodies from animals immunized with said protein. The potent accessory function of dendritic cells provides for an antigen presentation system for virtually any antigenic epitope which T lymphocytes are capable of recognizing through their specific receptors. Example 1, infra, demonstrates how dendritic cells can present viral antigen after acquisition from donor cells undergoing apoptosis, and effectively activate CTL responses.
Sources of Dendritic Cells
The dendritic cells used in this invention can be isolated as described herein or by methods known to those skilled in the art. In a preferred embodiment, human dendritic cells are used from an appropriate tissue source, preferably blood or bone marrow.
Mature dendritic cells can also be obtained by culturing proliferating or non-proliferating dendritic cell precursors in a culture medium containing factors which promote maturation of immature dendritic cells to mature dendritic cells. Steinman et al. U.S. Pat. No. 5,851,756 and U.S. applications application Ser. No. 08/600,483 (WO 97/29182) report methods and compositions for obtaining dendritic cells and are incorporated herein by reference.
The dendritic cell precursors, from which the immature dendritic cells for use in this invention are derived, are present in blood as PMBCs. Although most easily obtainable from blood, the precursor cells may also be obtained from any tissue in which they reside, including bone marrow and spleen tissue. When cultured in the presence of cytokines such as a combination of GM-CSF and IL-4 or IL-13 as described below, the non-proliferating precursor cells give rise to immature dendritic cells for use in this invention.
Culture of Pluripotential PMBCs to Produce Immature Dendritic Cells
Dendritic cell development can be divided into 4 stages: 1) a proliferating progenitor that can be either dendritic cell committed or uncommitted and capable of maturing to a nondendritic cell, 2) a non-proliferating precursor like the blood monocyte that does not show dendritic cell properties but is the starting population for many clinical studies, 3) an immature dendritic cell which has properties and commitment to become a dendritic cell, e.g. specialized antigen capture mechanisms including apoptotic cells for presentation, and MHC rich compartments, and 4) finally, the mature T cell stimulatory dendritic cell.
Cultures of immature dendritic cells, i.e. antigen-capturing phagocytic dendritic cells, may be obtained by culturing the non-proliferating precursor cells in the presence of cytokines which promote their differentiation. A combination of GM-CSF and IL-4 at a concentration of each at between about 200 to about 2000 U/ml, more preferably between about 500 and 1000 U/ml, and most preferably about 800 U/ml (GM-CSF) and 1000 U/ml (IL-4) produces significant quantities of the immature, i.e. antigen-capturing phagocytic dendritic cells, dendritic cells. Other cytokines or methods known in the art which efficiently generate immature dendritic cells may be used for purposes of this invention. Examples of other cytokines which promote differentiation of precursor cells into immature dendritic cells include, but are not limited to, IL-13. Maturation of dendritic cells requires the addition to the cell environment, preferably the culture medium,of a dendritic cell maturation factor which may be selected from monocyte conditioned medium and/or factors including TNF-α, IL-6, IFN-α, and IL-1-β. Alternatively, a mixture of necrotic cells or necrotic cell lysate may be added to induce maturation.
Co-culture of Dendritic Cells with Apoptotic Cells
Apoptotic cells may be used to deliver antigen to either immature or mature dendritic cells, either freshly isolated or obtained from in vitro culture. In a preferred embodiment, apoptotic cells comprising an antigen are co-cultured with immature dendritic cells for a time sufficient to allow the antigen to be internalized by the immature dendritic cells. These immature dendritic cells are then caused to mature by the addition of a maturation factor to the culture medium. The matured dendritic cells expressing processed antigen on their surface are then exposed to T cells for potent CTL induction.
In another embodiment, apoptotic cells may be used to deliver antigen to mature dendritic cells through surface receptors that enhance internalization of the apoptotic cells.
In another embodiment, apoptotic cells may be used to deliver antigen to immature dendritic cells that are maintained as immature dendritic cells as a means of efficiently modulating T cell tolerance or immunity in situ.
In a preferred embodiment, peripheral blood mononuclear cells [PBMCs] can be isolated from blood by sedimentation techniques. T cell-enriched [ER+] and T cell-depleted [ER−] populations can be prepared by resetting with neuraminidase treated sheep red blood cells. Dendritic cells are prepared from the ER− cells (Steinman et al. U.S. application Ser. No. 08/600,483) as discussed above and are preferably cultured for 7 days to 10 days in the presence of GM-CSF and IL-4. On about day 7 through 10, apoptotic cells can be co-cultured with the dendritic cells and the dendritic cells caused to mature over the next four days with the addition of monocyte conditioned medium, a signal for maturation.
Besides monocyte conditioned medium, a combination of cytokines may be used to induce maturation of the immature dendritic cells. Examples of cytokines which may be used alone or in combination with each other include, but are not limited to, TNF-α, IL-1β, IL-6, IFN-α and necrotic cells.
The apoptotic cell-activated dendritic cells made according to the method described above are the most efficient for induction of CTL responses. Delivery of antigen to mature dendritic cells, or alternatively, immature dendritic cells that are not caused to mature in vitro, is also within the scope of this invention.
The apoptotic cells useful for practicing the method of this invention should efficiently trigger antigen internalization by dendritic cells, and once internalized, facilitate translocation of the antigen to the appropriate antigen processing compartment.
In a preferred embodiment, the apoptotic cells, or fragments, blebs or bodies thereof, are internalized by the dendritic cells and targeted to an MHC class I processing compartment for activation of class I-restricted CD8+ cytotoxic T cells.
In another embodiment, the apoptotic cells can be used to activate class II-restricted CD4+ T helper cells by targeting antigen via the exogenous pathway and charging MHC class II molecules. Apoptotic cells, blebs and bodies are acquired by dendritic cells by phagocytosis. When a population of CD4+ cells is co-cultured with apoptotic cell-primed dendritic cells, the CD4+ T cells are activated by dendritic cells that have charged their MHC class II molecules with antigenic peptides. The apoptotic cell-charged dendritic cells of this invention activate antigen-specific CD4+ T cells with high efficiency.
For purposes of this invention, any cell type which expresses antigen and is capable of undergoing apoptosis can potentially serve as a donor cell for antigen delivery to the potent dendritic cell system. Examples of antigen that can be delivered to dendritic cells by donor cells include, but are not limited to, viral, bacterial, protozoan, microbial and tumor antigens as well as self-antigens. Preferred antigens for priming dendritic cells in vitro or in vivo are derived from influenza virus, malaria, HIV, EBV human papilloma virus (including both EBV-associated and EBV-unassociated lymphomas), CMV, renal cell carcinoma antigens, and melanoma antigens. In addition, self antigens that are targets of autoimmune responses can be delivered to dendritic cells e.g. insulin, histones, GAD.
For purposes of this invention the population of donor cells expressing antigen can be induced to undergo apoptosis in vitro or in vivo using a variety of methods known in the art including, but not limited to, viral infection, irradiation with ultraviolet light., gamma radiation, cytokines or by depriving donor cells of nutrients in the cell culture medium. Time course studies can establish incubation periods sufficient for optimal induction of apoptosis in a population of donor cells. For example, monocytes infected with influenza virus begin to express early markers for apoptotis by 6 hours after infection. Examples of specific markers for apoptosis include Annexin V, TUNEL+ cells, DNA laddering and uptake of propidium iodide.
Those skilled in the art will recognize that optimal timing for apoptosis will vary depending on the donor cells and the technique employed for inducing apoptosis. Cell death can be assayed by a variety of methods known in the art including, but not limited to, fluorescence staining of early markers for apoptosis, and determination of percent apoptotic cells by standard cell sorting techniques.
In one embodiment, donor cells are induced to undergo apoptosis by irradiation with ultraviolet light. Depending on the cell type, typically exposure to UV light (60 mjules/cm2/sec) for 1 to 10 minutes induces apoptosis. This technique can be applied to any cell type, and may be most suitable for a wide range of therapeutic applications. The apoptotic donor cells expressing an antigen of interest on their surface could then be used to prime dendritic cells in vitro or in vivo.
In another embodiment, donor cells are induced to undergo apoptosis by administering a drug such as which induces apopotosis. This technique can also be applied to any cell type, and is also suitable for a wide range of therapeutic applications.
In another embodiment, donor cells are induced to undergo apoptosis by infection with influenza virus. These apoptotic cells which express viral antigens on their surface could then be used to prime dendritic cells in vitro or in vivo. The apoptotic cell-activated dendritic cells may then be used to activate potent influenza-specific T cells.
In another embodiment, tumor cells may be obtained and caused to undergo apoptosis. These apoptotic tumor cells, or tumor cell lines, could then be used to deliver tumor antigen to dendritic cells in vitro or in vivo. Once isolated, the tumor cells could be treated with collagenase or other enzymes which facilitate cell dissociation for culturing. The apoptotic cell-activated dendritic cells may then be used as cancer therapeutic agents by activating the immune system to specifically target the tumor cells.
In another embodiment of the invention, the donor cells can be transfected, transduced or transformed to express foreign antigens prior to induction of apoptosis. In this manner dendritic cells may be loaded with antigens not typically expressed on the donor cell. In addition, delivery of antigens via xenotransfer is also contemplated. These methods can be accomplished using standard techniques known in the art.
A variety of possible antigens can be used in this invention including, but not limited to, bacterial, parasitic, fungal, viral, and tumor antigens of cellular or viral origin. Preferred antigens include influenza virus, malaria, HIV, EBV, human papilloma virus, CMV, renal cell carcinoma antigens, and melanoma antigens. In addition, self antigens that are targets of autoimmune responses or other antigens for which it is desired to attenuate an immune response can be expressed on donor cells using any of the aforementioned methods.
Once donor cells expressing either native or foreign antigen have been induced to undergo apoptosis they can be contacted with an approprite number of dendritic cells in vitro or in vivo. The ratio of apoptotic cells to dendritic cells may be determined based on the methods disclosed in Example 1 or Example 6, infra. For most antigens a ratio of only about 1–10 donor cells to 100 immature dendritic cells is suitable for priming the dendritic cells. Higher numbers of apoptotic cells are preferred if mature dendritic cells are to be primed, preferably 100 to 1000 donor cells to mature dendritic cells.
The population of apoptotic cells should be exposed to the dendritic cells for a period of time sufficient for the dendritic cells to internalize the apoptotic cell, or apoptotic cell fragments. Efficiency of cross-priming or cross-tolerizing dendritic cells can be determined by assaying T cell cytolytic activity in vitro or using dendritic cells as targets of CTLs. Other methods known to those skilled in the art may be used to detect the presence of antigen on the dendritic cell surface following their exposure to apoptotic donor cells. Moreover, those skilled in the art will recognize that the length of time necessary for an antigen presenting cell to phagocytose apoptotic cells, or cell fragments, may vary depending on the cell types and antigens used.
An important feature of the dendritic cells of this invention is the capacity to efficiently present antigens on both MHC class I and class II molecules. Apoptotic donor cells, blebs, bodies or fragments thereof, are acquired by dendritic cells through the exogenous pathway by phagocytosis and as a result also efficiently charge MHC II molecules. CD4+ T cells may be activated by the dendritic cells presenting antigenic peptide which is complexed with MHC II using the method according to this invention, since it is known in the art that dendritic cells are the most potent inducers of CD4+ helper T cell immunity. CD4+ T cells can provide critical sources of help, both for generating active CD8+ and other killer T cells during the acute response to antigen, and for generating the memory that is required for long term resistance and vaccination. Thus, by using apoptotic cells to charge MHC class I and/or II products, efficient T cell modulation in situ can be achieved.
In a preferred embodiment both apoptotic and necrotic donor cells are used to prime dendritic cells. Preferably immature dendritic cells are contacted with the donor cells and both class I and II MHC receptors become optimally charged with antigen. In addition, the dendritic cells are matured by the presence of the necrotic cells which also contributes to MHC class II loading.
A novel aspect of this invention is the use of apoptotic cells to prime dendritic cells with antigen. An important advantage of the apoptotic cell system is that even poorly defined or undefined antigens can be routed to the appropriate antigen processing compartment of the dendritic cells to generate antigen-specific T cell responses. Moreover, dendritic cells can be charged with multiple antigens on multiple MHCs to yield poly or oligoclonal stimulation of T cells. Thus, since the starting material can be obtained from virtually any tissue, the only requirement for efficient targeting of potentially any antigen to dendritic cells is the packaging of the antigen with apoptotic cells or apoptotic cell material.
Accordingly, the scope of this invention includes an embodiment whereby the apoptotic cell delivery system is reconstituted in vitro using apoptotic cell fragments, or material thereof, including, but not limit to, proteins, phospholipids, carbohydrates and/or glycolipids, which enhance internalization and translocation of antigen to the appropriate antigen processing compartment in the dendritic cells. Thus, it is contemplated that liposomes comprising at least antigen and any combination of the aforementioned apoptotic cell fragments, or materials thereof, may enhance delivery of antigen to dendritic cells. Liposomes enhanced with apoptotic cell fragments, or materials thereof in accordance with this invention, can also be used for delivering antigen in vivo to dendritic cells.
As stated above, to prepare the liposomes it is desirable to include ligands for various receptors on the dendritic cells including various integrin receptors, CD36 and heat shock proteins. It is also contemplated that various mutant forms or fragments containing binding domains of such proteins may also be used. If DC are pulsed with liposomes in vitro, between 0.1–1000 ng of antigen is preferred for priming between 1×105 and 5 million DCs. Higher amounts of antigen in liposomes are used to deliver between about 10 ng to 10 μg per immunization if DCs are to be pulsed in vivo.
In another embodiment, the apoptotic cell antigen delivery system can be used as a method for identifying and isolating poorly characterized antigens. For example, a population of tumor cells can be induced to undergo apoptosis, the apoptotic tumor cells can then be co-cultured with dendritic cells for a time sufficient to allow relevant immunogenic peptides to be processed and presented on the dendritic cell surface. The immunogenic peptides can then be eluted from the MHC and other presenting molecules of dendritic cell surface as disclosed in U.S. Pat. No. 5,851,756 and purified using standard protein purification techniques known in the art including, but not limited to, HPLC and mass spectrophotemetry.
Activation of Dendritic Cells and CTLs
In yet another embodiment of this invention, the apoptotic cell-charged dendritic cells or the antigen-specific T lymphocytes generated by methods described herein may be used for either a prophylactic or therapeutic purpose. For activating T cells in an individual between about 2×105 and 2×109 more preferably between 1 million and 10 million apoptotic cell-activated mature dendritic cells should be administered to an individual. The dendritic cells should be administered in a physiologically compatible carrier which is nontoxic to the cells and the individual. Such a carrier may be the growth medium described above, or any suitable buffering medium such as phosphate buffered saline (PBS). The mature dendritic cells prepared according to this invention are particularly potent at activating T cells. For example, using prior methods of dendritic cells the ratio of dendritic cells to T cells necessary for strong T cell activation is about 1 dendritic cell to 30 T cells whereas the ratio according to this invention is about 1 T cell to 1000 dendritic cells. Thus, fewer dendritic cells are required. For activating T cells in vitro the ratio of dendritic cells to T cells is between 1:10 and 1:1000. More preferably, between 1:30 and 1:150. Between approximately 106 and 109 or more activated T cells are administered back to the individual to produce a response against the antigen.
Dendritic cells may be administered to an individual using standard methods including intravenous, intraperitoneal, subcutaneously, intradermally or intramuscularly. The homing ability of the dendritic cells facilitates their ability to find T cells and cause their activation.
By adapting the system described herein, dendritic cells could also be used for generating large numbers of CD8+ CTL, for adoptive transfer to immunosuppressed individuals who are unable to mount normal immune responses. Immunotherapy with CD8+ CTL has been shown to amplify the immune response. Bone marrow transplant recipients given CMV specific CTL by adoptive transfer, do not develop disease or viremia (4). These novel approaches for vaccine design and prophylaxis should be applicable to several situations where CD8+ CTLs are believed to play a therapeutic role e.g. HIV infection (1–3), malaria (5) and malignancies such as melanoma (8–9).
Examples of diseases that may be treated by the methods disclosed herein include, but are not limited to bacterial infections, protozoan, such as malaria, listeriosis, microbial infections, viral infections such as HIV or influenza, cancers or malignancies such as melanoma, autoimmune diseases such as psoriasis and ankolysing spondylitis.
By adapting the system described herein, dendritic cells could also be used for inducing tolerance. Dendritic cells expressing fas-L for example can kill fas expressing activated T cells (23). Thus, it may be possible to deliver fas-L bearing dendritic cells that are pulsed with apoptotic cells or blebs or bodies containing autoantigens to delete autoreactive T cells in vivo; or alternatively, one can target dendritic cells that are specialized to induce tolerance rather than immunity, as proposed for the subset of dendritic cells known as “lymphoid dendritic cells” (see Steinman et al., Immunol. Rev. 156: 25–37, 1997).
Methods for Assessing Cytotoxic T Cell Activity
Frequently, in clinical disease it is difficult to detect killer cells because of inadequate presentation. This invention also provides methods for assessing the cytotoxic activity of T lymphocytes, and in particular the ability of cytotoxic T lymphocytes to be induced by antigen presenting dendritic cells. According to this method, a sample comprising T lymphocytes to be assayed for cytotoxic activity is obtained. Preferably, the cells are obtained from an individual from whom it is desirable to assess their capacity to provoke a cytotoxic T lymphocyte response. The T lymphocytes are then exposed to antigen presenting dendritic cells which have been caused to present antigen. Preferably, the dendritic cells have been primed with apoptotic cells which express antigen. After an appropriate period of time, which may be determined by assessing the cytotoxic activity of a control population of T lymphocytes which are known to be capable of being induced to become cytotoxic cells, the T lymphocytes to be assessed are tested for cytotoxic activity in a standard cytotoxic assay. Such assays may include, but are not limited to, the chromium release assay described herein.
The method of assessing cytotoxic T lymphocyte activity is particularly useful for evaluating an individual's capacity to generate a cytotoxic response against cells expressing tumor or viral antigens. Accordingly, this method may be useful for evaluating an individual's ability to defend against cancers, for example melanoma, or viruses. In addition, this method is useful to detect autoreactive killer cells and could monitor not only the presence of killer cells, but their response to therapy.
All books, articles, or patents referenced herein are incorporated by reference in their entirety.
Generation of mononuclear subsets. Peripheral blood mononuclear cells [PBMCs] were isolated from blood by sedimentation in Ficoll-Hypaque [Pharmacia Biotech]. T cell-enriched [ER+] and T cell-depleted [ER−] populations were prepared by resetting with neuraminidase treated sheep red blood cells, as previously described (37). T cells were purified from ER+ cells by removal of monocytes, NK cells and MHC class II+ cells (37). Monocytes were obtained from ER− cells by plastic adherence. Dendritic cells were prepared from ER− cells cultured for 7 days in the presence of GM-CSF and IL-4, followed by 4 days in monocyte conditioned medium (42, 43).
Influenza A virus establishes a nontoxic infection in human DCs (37,38, 2016–4008). Once infected, these DCs are capable of eliciting virus-specific recall CTL responses within 7 days. The CTLs generated are class I-restricted and kill virus-infected monocytes and peptide pulsed target cells (37,39). Compared with DCs, influenza infected monocytes are poor stimulators of CTL responses (37) and undergo apoptosis (40,41). We have exploited these observations to investigate the role of apoptosis in the generation of antigen which could be acquired by uninfected DCs.
DCs were prepared from peripheral blood precursors of HLA-A2.1+ individuals (42,43). Uninfected DCs and syngeneic T cells were co-cultured with influenza-infected syngeneic [
To confirm that antigen is transferred to, and expressed directly by, DC MHC class I products, we co-cultured uninfected DCs and influenza-infected monocytes, and used the DCs as targets for CTLs [
Given the nature of the antigen, it was important to exclude live virus as the agent responsible for the transfer of antigen to DCs. Based on previous studies (14,20), influenza-infected monocytes produce only low levels of infectious virus before they undergo apoptosis. Infection of 5×104 monocytes with an MOI of 2.0 would be expected to yield up to 1×103 infectious virions after a 24 hour culture period. To prevent infection of DCs by monocyte produced virus, all experiments were done in human serum. The presence of blocking anti-hemagglutinin antibodies prevents influenza virions from binding to cell surface glycoconjugates. Indeed, the addition of influenza virus [highest dose, 104 infectious virions] directly into cultures containing uninfected DCs and T cells did not generate a CTL response [data not shown]. To further exclude the possibility that monocytes were releasing virus during the 7 day co-culture, we tested for the presence of free virions using a standard hemagglutination assay with chicken RBC (45). Less than 1 HAU/ml [hemagglutination units per milliliter] was detected in the medium at 12 hr, 24 hr and 7 days of co-culture.
We also exclude the possibility that the CTL responses were due to free peptide being released by the dying monocytes, thereby charging class I molecules on DCs. Media from wells containing the infected monocytes were collected after an overnight culture and transferred to fresh wells containing T cells and uninfected DCs. Virus-specific CTLs were generated after a 7 day culture period [
The role of apoptosis in the transfer of antigen to the uninfected DC was therefore assessed. We first show that influenza-infected monocytes undergo apoptosis as detected by annexin V binding, an early marker for programmed cell death [
To establish that apoptosis is the trigger for cross-priming, we first used Z-VAD-CHO, irreversible peptide inhibitor of caspase activity [Kamiya Biomedical Company]. It was possible to block apoptosis of influenza-infected monocytes [determined by TUNEL], without affecting the expression of viral proteins, [evaluated by staining with anti-nucleoprotein antibodies, data not shown]. Influenza-infected monocytes were cultured overnight in the presence of varying concentrations of Z-VAD. The medium from these wells were then added to fresh wells containing T cells and uninfected DCs. After 7 days, the responding T cells were measured for their influenza-specific cytolytic activity. At concentrations of Z-VAD which inhibited apoptosis of the infected monocytes, antigenic material was not transferred to the uninfected DCs and virus-specific CTLs were not generated [
We next compared apoptotic death to necrotic death for the generation of antigenic material. Influenza-infected 293 cells were used for this experiment. Greater than 95% of the cells expressed influenza proteins after 10 hours, but unlike monocytes, apoptosis was not triggered. Apoptosis was induced in the 293 cells by exposure to 60 mJ/cm2 UVB irradiation (47), and necrosis was achieved by incubation in a hypotonic solution for 30 minutes at 37° C. Apoptotic or necrotic cells were co-cultured with uninfected DCs and T cells for 7 days and responding T cells were tested as described above. Only apoptotic but not necrotic cells were capable of transferring antigen to the DCs, as determined by the generation of a potent influenza-specific CTL response [
The cellular requirements for generating CTLs via this pathway were also investigated. Given that lysis of T2 cells, an HLA-A2.1+ cell line, was dependent on the matrix peptide, which is specific for HLA-A2.1, it was expected that the effectors were MHC class I-restricted [
We next examined whether co-cultures of uninfected DCs, influenza-infected monocytes and purified CD8+ T cells could generate CTLs [
DCs and macrophages were compared as potential mediators of this exogenous pathway for class I MHC presentation. Uninfected HLA-A2.1+ DCs or macrophages, and syngeneic T cells were co-cultured with infected HLA-A2.1− monocytes. After 7 days, the responding T cells were measured for their influenza-specific cytolytic activity. DCs, but not macrophages, were capable of stimulating influenza-specific CTLs [
Different pathways have been proposed for the presentation of exogenous antigen on MHC class I molecules. One possibility is that the antigens access the cytoplasm directly from the endosomal compartments and enter the ‘classical’ class I processing pathway (26,29). Alternatively, class I molecules associated with invariant chain may enter the endosomes (49). Finally, peptides generated in the endosomes may be regurgitated to the cell surface, charging class I molecules (34). We investigated if intracellular processing was required for DCs to present antigens from apoptotic cells. Uninfected DCs pulsed with influenza-infected allogeneic monocytes were used as targets for influenza specific CTLs [Table I]. A 3–9 hr co-culture of uninfected DCs and infected apoptotic monocytes was sufficient to charge DCs with antigen. The DCs were preincubated with 10 mM ammonium chloride, which neutralizes the endocytic compartment, or 6 ug/ml Brefeldin A [BFA], an inhibitor of vesicular transport (50). Both ammonium chloride and BFA completely inhibited the DCs ability to present antigenic material on MHC class I. Lactacystin, an irreversible inhibitor of the 26S proteasome (51), only partially blocked antigen presentation by the uninfected DCs [unpublished data]. The data suggest that both classical and non-classical class I pathways are utilized for the presentation of exogenous antigen derived from apoptotic cells.
Since processing was required for presentation of monocyte derived antigen, immunofluorescence and electron microscopy were used to document that DCs phagocytosed apoptotic cells. By immunofluorescence, we observed phagocytosis of fragmented or intact cell bodies in 10–20% of the DCs [
Previous studies have shown that murine DCs have the capacity to present soluble antigens via an exogenous pathway, leading to the induction of MHC class I-restricted CTLs (32, 53–55). In addition, a single report reported that following intra-venous injection of allogeneic cells, rat interdigitating DCs within lymphnodes were found to contain whole cells and cell fragments (56). Using virus-infected monocytes, which themselves do not induce CTLs, we show that human DCs can acquire physiologically relevant antigens by phagocytosing apoptotic cells and then stimulating MHC class I-restricted CTLs.
The methods and materials unless otherwise specified were the same as in Example 1.
The following methods were used to carry out the experiments shown in
The results demonstrate that a transfected tumor cell line can serve as a donor apoptotic cell, allowing for the transfer of antigen to the uninfected dendritic cell and the effective induction of antigen-specific CD8+ CTLs. Controls included: mock transfected cells (open triangles) infected and uninfected 293 cells (filled and open circles, respectively), infected 293 cells without DCs (filled circles, dotted line), infected and uninfected DCs (filled and open diamonds, respectively).
The following methods were used to carry out the experiments shown in
Given that the lysis of T2 cells, an HLA-A2.1+ cell line, was dependent on the matrix peptide, which is specific for HLA-A2.1, it was expected that the effectors were MHC class I-restricted (see Example 1). To confirm this, highly purified CD4+ and CD8+ subpopulations were isolated at the end of a 7 day culture period. As demonstrated in
The following methods were used to carry out the experiments shown in
Immature DCs pulsed with apoptotic influenza infected EL4 cells in the context of monocyte conditioned media, were used to stimulate bulk T cells. The immature DCs efficiently engulf the murine tumor cell line and are able to cross-present influenza antigen derived from those apoptotic cells. Indeed, at a ratio of DCs:EL4s equal to 100:1, it was still possible to elicit potent T cells responses. This experiment also demonstrates the possibility of using xenotropic cells as a source of antigen bearing apoptotic cells for the delivery of antigenic material to the DCs.
The following methods were used to carry out the experiments shown in
Immature DCs but not Mature DCs or Monocytes cross-present antigenic material from apoptotic cells and become targets of antigen-specific CTLs. [
To better define the antigen presenting cell capable of cross-presentation of antigenic material derived from the engulfment of apoptotic cells, an assay was established in which the APC is used as a target for antigen-specific CTLs in a standard chromium release assay. Various populations of HLA-A2.1+ professional APCs were co-cultured with HLA-A2.1− influenza infected monocytes. After 12 hrs, the APCs were loaded with 51Cr and used as targets for an influenza-reactive CTL line. Specific lysis indicates that the APC had cross-presented antigenic material derived from the apoptotic cell, leading to the presence of specific peptide-MHC class I complexes on its surface.
While mature DCs were capable of serving as targets when infected with influenza, they were unable to engulf apoptotic cells presumably because the mechanisms of antigen uptake, phagocytosis in this case, had been down regulated. Immature DCs co-cultured with apoptotic allogeneic influenza-infected monocytes were capable of serving as targets. When cultured in the presence of conditioned media, a maturation signal for the DC, these cells upregulate costimulator molecules which apparently permit the DCs to serve as more efficient targets. Notably, neither the mature or the immature macrophages cross-presented antigenic material derived from the apoptotic cells. Unlike the mature DC population (which also do not serve as effective targets in this assay), these APCs are capable of phagocytosing apoptotic cells, however the engulfed material is most likely degraded and not cross-presented on MHC class I. When the various HLA-A2.1+ APC populations were infected with influenza, all served as effective targets demonstrating that the differences described above were not a function of MHC class I down-regulation or an inability for presentation of antigen.
The HLA-A2.1− monocytes which were used as a source of apoptotic cells were also tested as targets for the influenza-specific CTLs in order to confirm that the observed lysis was not due to killing of the allogenic influenza-infected monocytes. This result suggests that in vivo, it is the immature DC which is responsible for the uptake of apoptotic cells. After maturation and migration to a T cell rich area of a primary or secondary lymph organ, the DCs could stimulate CD8+ class-I restricted CTLs. Also indicated by this result is that the preferable method for immunotherapy would be to first co-culture immature DCs with apoptotic cells (a source of exogenous antigenic material) along with a maturation signal (CM, TNF α, IL1 β, IFN −α, or some combination of the aforementioned) prior to injection.
Mice. Adult 6–8 week mice [BALB/C, C57BL/6, BALB/C×DBA2 F1, C57BL/6×DBA/2 F1] of both sexes were purchased from Taconic Farms [NY] and Japan SLC [Hamamatsu, Japan].
Cells. DCs were generated from bone marrow progenitors by culture in rGM-CSF as described (86). The cultures were set up in 24 well plates [Costar, Cambridge Mass.; Nunc, Naperville Ill.] and used at d6 when the wells were covered with aggregates of immature DCs. By d7-8, aggregates release mature DCs, about 105/well (86). Immature but not mature DCs are phagocytic for several particulates (79). B blasts were induced with lipopolysaccharide [E.coli 0111:B4, Sigma Chemical, St. Louis Mo., 10, μg/ml] for 3–4 d, or with anti-μ [Jackson Immunoresearch Labs, West Grove Pa.; F[ab] ′2 goat anti-mouse IgM] and IL-4 [Gibco-BRL] for 2 d. B cells were spleen cells that were passed over Sephadex G10 columns and depleted of T cells with antibodies and complement [thy-1, TIB 99; CD4, L3T4; CD8, TIB 211; all from ATCC]. In some cases, necrosis was induced in the B cells either by freeze-thawing 3–4 times, or with anti-B220 antibody [monoclonal J11d] and rabbit complement. Human cells were plastic adherent, monocytes from peripheral blood mononuclear cells and EBV-transformed B cell lines. Apoptosis was induced by infection with influenza (95), or by exposure to LPS [10 ng/ml] or UV light.
Antibodies. The Y-Ae hybridoma was generously provided by Dr. C. Janeway, Yale University, New Haven Conn. and grown serum-free. The IgG2b antibody was purified on protein A sepharose, biotinylated, and used in the FACS along with PE-streptavidin to detect MHC class II-peptide complexes (75,76) with a mouse IgG2b anti-human TCR antibody as the non-reactive control [PharMingen, San Diego Calif.]. The cells were double labeled for FACS and for microscopy with FITC antibodies to CD8, CD11c, CD86, I-Ab [AF6-120.1], 1-Ad [39-10-8], and I-E [14-4-4S] [PharMingen].
Capture of cell-derived peptides in vitro. Bone marrow DCs were generated in 24 well plates for 6 days in GM-CSF, gently washed to remove mature floating DCs, and then fed B blasts, human cells, or a peptide [residues 56–73 of I-Eα]. Before adding I-E+ cells or peptide, the DCs were immature, since much of the MHC class II was in intracellular lysosomal compartments and CD86 was not expressed at the surface (84) After uptake of I-E, the DCs matured with high levels of surface MHC II and CD86.
Antigen presentation assays. The 20.6 T-T hybridoma recognizes the MHC II-peptide complex that is detected with Y-Ae mAb (81). Graded doses of APCs were added to 2×104 hybridoma cells in flat bottomed microtest plates. At 24 h, supernatants were assayed for IL2 using a growth assay for conA stimulated T blasts.
Capture of cell-derived peptides in vivo. 2×106 H-2d BALB/C DCs were injected in PBS into the footpads of C57BL/6 [H-2b] or as a negative control, CBA/J [H-2k] mice. After 2 days, the draining lymph nodes were removed, frozen in OCT embedding medium, sectioned, fixed in absolute acetone 5 min, and stained successively with biotin-Y-Ae, avidin alkaline phosphatase conjugates, B220 for B cells, and peroxidase labeled mouse anti-rat Ig. We then could visualize the position of blue Y-Ae+ cells relative to B cells which are found in white pulp follicles and scattered through the red pulp. To identify the Y-Ae+ cells, DCs were enriched from the nodes as described 25 and double labeled for biotin Y-Ae and PE streptavidin [or nonreactive biotin IgG2b mAbas control] and different FITC conjugates to CD8, B220, CD11c, I-Ab, I-Ad.
The development of many immune responses requires that antigen presenting cells [APCs] present antigens that initially are expressed by other cells. This antigen transfer to APCs may often be essential, since most cell types lack the migratory and costimulatory properties required to initiate a T cell response. Examples of responses that can develop following antigen transfer, often termed cross-priming and cross-tolerance, include the rejection of malignant cells and transplants, including xenografts (64–70), and the maintenance of tolerance to self tissues (71–73). However, underlying mechanisms have been difficult to identify directly. Prior experimental approaches have utilized hematopoietic chimeras. In the chimera, the T cells that become primed to transplanted or malignant tissue [or tolerized to self] recognize antigen presenting MHC products on bone marrow-derived cells. It is possible that antigen transfer simply involves the release of peptides from donor [tumor, transplant, self tissue] to recipient APCs. We have shown in the previous examples how human dendritic cells [DCs] present viral antigens from apoptotic infected cells to MHC class I restricted, cytolytic T lymphocytes (74). In this example, we have used an antibody to directly monitor the formation of specific MHC class II peptide complexes when DCs [which are bone marrow-derived] are exposed to other noninfected cells. In vitro, we find that antigen transfer requires phagocytosis, takes place with both xenogeneic and transformed cells. In vivo, where antigen transfer can be visualized with the antibody to MHC-peptide, DCs serve as a powerful donor and recipient of cellular peptides. We propose that the steady state migration of DCs in afferent lymph, followed by processing by resident DCs in the T cell areas of lymphoid tissues, continuously tolerizes the T cell repertoire to self.
To demonstrate the capacity of DC to present peptides from other cells, we took advantage of the Y-Ae monoclonal antibody. Y-Ae sees an MHC class II-peptide complex that is formed when one MHC II product, I-Ab, presents a peptide from residues 56–73 of another MHC II product, I-Eα (75,76). Actually, the repertoire of peptides presented on MHC products typically include other MHC-derived peptides (77,78). We cultured C57BL/6 DCs [I-Eα−,I-Ab+] with BALB/C B cells [I-Eα+,I-Ab-−] and monitored the capacity of DCs to react with Y-Ae by cytofluorography. The DCs were from 6 day cultures of GM-CSF stimulated mouse bone marrow, since these are known to have phagocytic activity, in contrast to the mature nonphagocytic DCs that predominate by day 7–8 of these cultures (79). The B cells were activated 3–4 with LPS or with anti-α plus IL-4 to express high levels of MHC II (80). When returned to culture, 20–40% of the B blasts undergo apoptosis spontaneously, so that it is unnecessary to induce apoptosis in these I-E rich cells, in contrast to human monocytes (below) where the need for apoptosis is shown for transfer of cell-associated antigens to DCs.
The MHC II-peptide complexes that formed during antigen transfer from B blasts to DCs could be recognized by T cells, as shown with a hybridoma that responds to the I-Ab/Eα peptide complex by secreting IL-2 (81). Following a 20 hour exposure to I-Eα peptide or B blasts, the DCs were sorted as CDllc/CD86+ cells, fixed in formaldehyde to block further processing, added in graded doses to the T-T hybridoma for 24 hrs, and then the medium collected for testing in an IL-2 bioassay [3H-TdR uptake,
To rule out the possibility that B blasts were simply releasing peptide to the DCs, we showed that Y-Ae labeling did not develop if B blasts were separated from DCs by a Transwell filter [0.45μ pores], or if B blasts were added to nonphagocytic DCs that had undergone maturation in the bone marrow culture [
Since the immune response to xenografts also involves processing of donor cells by host APCs (70), we tested human cells as the donor of peptides to DCs. This was feasible with the Y-Ae monoclonal, since human HLA-DRα chain [residues] has the identical sequence to mouse I-Eα 56–73 (82–83). The Y-Ae epitope developed when mouse DCs were cultured with either EBV-transformed, human B lymphoblastoid cell lines [
To demonstrate that phagocytosis preceded the formation of the Y-Ae epitope, B blasts were fed to DCs in 20 mM NH4Cl. After 20 h, DCs were FACS® separated from residual B blasts and recultured for 12 h without NH4Cl. NH4Cl completely blocked Y-Ae epitope development [FIG. 12A, top], even within saponin-permeabilized cells (not shown), but the Y-Ae reappeared after removal of NH4Cl [
To determine whether this mechanism could function in vivo, we injected different I-Ab− I-E+ cell types [mouse splenocytes, B blasts and DCs; human monocytes] into the footpads of CS7BL/6 [I-Ab+ I-E−] mice and then examined draining lymph nodes 2 days later for Y-Ae+ cells. The negative controls were H-2k C3H recipients. Only mature DCs, derived from mouse bone marrow cultures (24), were found to be efficient donors of I-E peptide to MHC II of host cells, with numerous Y-Ae+ cells developing in the DC-rich, T cell areas [
Many immune responses require that APCs present antigens from other cells (63–73). Most cells in the body do not migrate to the T cell areas of lymphoid tissues and lack costimulatory functions [like CD86], both properties being important for selecting and activating T cell-mediated responses. In contrast, DCs are a potent system for stimulating T cell immunity and tolerance, having the required migratory and stimulatory functions (91). This data, together with data on the presentation of apoptotic, virus-infected cells on MHC class I (74) outline a new function for DCs, the presentation of antigens from other cells.
The consequences of antigen transfer to DCs, i.e., immunity [cross-priming] vs. nonresponsiveness [cross tolerance], may be influenced by stimuli in the DC environment. When DCs capture antigens from dying cells at a site of foreign antigen deposition, e.g., when microbial agents infect and kill somatic cells, or when transplants undergo rejection by what is termed the indirect pathway, the DCs may be altered by an inflammatory stimulus to be immunogenic. On the other hand, unresponsiveness or cross tolerance may develop when DCs capture antigens chronically from the normal, noninflammatory, apoptotic turnover of cells in vivo. Cross tolerance of self-reactive T cells is observed, for example, when antigens from β cells of normal, noninflammed pancreatic islets are presented by APCs in draining lymph nodes (72,73).
Substantial numbers of DCs traffic constitutively in the afferent lymph to the T cell areas where they almost certainly die, because the numbers of DCs in lymphoid tissues are normally steady and DCs do not emerge into the efferent lymph. Prior thinking has emphasized the potential value of this migratory, short lived, patrolling DC population to pick up foreign antigens and then present these in the T cell areas. Our results suggest that migratory DCs are also being processed distributing their peptides to other DCs in lymphoid organs. In our experiments, it is unlikely that Y-Ae+ cells were first formed in the periphery since B blasts, splenocytes, UV-treated DCs and human monocytes each did not lead to Y-Ae epitope formation in lymphoid organs. Instead, it appeared necessary for viable DCs to first migrate to the lymphoid organ, and then the vast majority of host, I-Ab, lymph node DCs captured I-E+ donor DCs to form the Y-Ae MHC-peptide complexes. The recipient Y-Ae+ cells in the T cell areas, sometimes termed “lymphoid DCs,” may be long-lived and are thought to have a regulatory function (90,92). In the steady state, i.e., in the absence of inflammatory signals, the continued processing of migratory DCs and any cellular antigens that they have acquired from the normal turnover of cells in tissues, could purge the T cell repertoire of self reactivity (72,73), a function that is also carried out on developing T cells by DCs in the thymus (93,94). As result, when foreign inflammatory stimuli are to be presented to the immune system by DCs, there is less chance that autoreactivity to self antigens develops, since the same DC system has inactivated self reactive cells beforehand.
Media. RPMI 1640 supplemented with 20 μg/ml of gentamicin [Gibco BRL], 10 mM HEPES [Cellgro] and either 1% human plasma, 5% pooled human serum [c-six diagnostics] or 5% single donor human serum was used for DC preparation, cell isolation and culture conditions (113,119).
Preparation of Cells. Peripheral blood mononuclear cells [PBMCs], DCs, macrophages and T cells were prepared as previously described (113,114,119). Briefly, peripheral blood was obtained from normal donors in heparinized syringes and PBMCs were isolated by sedimentation over Ficoll-Hypaque [Pharmacia Biotech]. T cell enriched and T cell depleted fractions were prepared by resetting with neuraminidase-treated sheep red blood cells (119). Immature dendritic cells [DCs] were prepared from the T cell depleted fraction by culturing cells in the presence of granulocyte and macrophage colony-stimulating factor [GM-CSF] and interleukin 4 [IL-4] for 7 days. 1000 U/ml of GM-CSF [Immunex Corp.] and 500–1000 U/ml of IL4 [Schering-Plough Corp.] were added to the cultures on days 0, 2 and 4. To generate mature DCs, the cultures were transferred to fresh wells on day 7 and monocyte conditioned media [MCM] was added for an additional 3–4 days (113,114). At day 7, >95% of the cells were CD14−, CD83−, HLA-DRlo DCs. On day 10–11, 80—100% of the cells were of the mature CD14−, CD83+, HLA-DRhi phenotype. FACSort® was used to generate highly pure populations of immature and mature DCs, based on their CD83− and CD83+ phenotype, respectively. Macrophages were isolated from T cell depleted fractions by plastic adherence for one hour. After 24 hrs., cells were removed from the plates and placed in Teflon beakers for 3–9 days. T cells were further purified from the T cell enriched fraction by removing contaminating monocytes, NK cells and B cells (119).
Antibodies. Antibodies to the following proteins were used: CD8-PE, CD14-PE, HLA-DR-PE, HLA-DR-biotin [Becton Dickinson], IgG2b [clone 6603001, Coulter], CD8 [CRL 8014, ATCC], CD83 [clone HB15a, Coulter], MHC I [W6/32, ATCC clone HB95], CD36 [clone FA6, obtained from the Vth international workshop on leukocyte differentiation antigens], αv [clone CLB-706, Chemicon International Inc.; clone 69.6.5, Coulter], β1 [clone 6S6, Chemicon International Inc.], β3 [clone SZ21, Coulter; clone RUU-PL 7F12, Becton Dickinson], β5 [clone B5-IVF2, Upstate biotechnology], αvβ3 [clone 23C6, Pharmingen], αvβ5 [clone P1F6, Chemicon International Inc.], CD71 [Dako], Mannose receptor [clone 3.2PB1, a gift from A. Lanzavecchia], Nucleoprotein [ATCC clone HB85].
Induction of apoptotic death. Monocytes were infected with influenza virus in serum free RPMI. These cells undergo viral induced apoptotic death within 6–8 hours. Cell death was confirmed using the Early Apoptosis Detection Kit [Kayima Biomedical] (102). As previously described, cells are stained with Annexin V-FITC [Ann V] and Propidium Iodide [PI]. Early apoptosis is defined by Ann V+/PI− staining as determined by FACScan® [Becton Dickinson]. To ensure that we were studying the uptake of early apoptotic cells, the kinetics of death were carefully worked out. 5–8 hours post-infection, monocytes first externalize PS on the outer leaflet of their cell membrane, as detected with Ann V. By 8–10 hours, these cells were TUNEL positive. It was not until 24–36 hours that the majority of the monocyte population included trypan blue into the cytoplasm, an indicator of secondary necrosis [unpublished data, (120,121)]. HeLa cells were triggered to undergo apoptosis using a 60UVB lamp [Derma Control Inc.], calibrated to provide 2 mJ/cm2/sec. The kinetics of cell death in these cells has been previously defined (99).
Phagocytosis of apoptotic cells. Monocytes or HeLa cells were dyed red using PKH26-GL [Sigma Biosciences], and induced to undergo apoptosis by influenza infection or UVB irradiation, respectively. After 6–8 hours, allowing time for the cells to undergo apoptosis, they were co-cultured with phagocytic cells that were dyed green using PKH67-GL [Sigma Biosciences], at a ratio of 1:1. Macrophages were used 3–6 days after isolation from peripheral blood; immature DCs were used on day 6–7 of culture; and mature DCs were used on day 10–11. Where direct comparison of cells was needed, cells were prepared from the same donor on different days. In blocking experiments, the immature DCs were pre-incubated in the presence of 50 μg/ml of various monoclonal antibodies for 30 minutes prior to the establishment of co-cultures. After 45–120 minutes, FACScan® analysis was performed and double positive cells were enumerated.
Phagocytosis of latex beads. Immature DCs were preincubated at 37° C. with monoclonal antibodies specific for αv and αvβ5. 106 cells were then cultured with 5×107 red fluorescent microspheres [diameter 1μ, 2.5% solids, carboxylate modified latex; Sigma] for varying periods of time. Alternatively, the cells were maintained 4° C. At the end of the assay, cells were separated from unengulfed beads by density gradient centrifugation and analyzed by FACScan® analysis (122).
Immunofluorescence. Cells were adhered to Alcian Blue [Sigma] treated cover slips and fixed in 100% acetone. Cells were stained with anti-influenza nucleoprotein antibody [HB85, ATCC] and Texas red conjugated goat anti-mouse IgG [Jackson ImmunoResearch]. This was followed by staining with biotinylated anti-HLA-DR [Becton Dickinson] followed by FITC conjugated streptavidin [Jackson ImmunoResearch]. Cells were visualized using an Zeiss Axioplan2 microscope.
Assay for cross-priming of apoptotic cells. Various APC populations were prepared as described above from HLA-A2.1+ donors. Mature DCs were further purified by labeling with the DC-restricted marker CD83, followed by cell sorting on the FACSort® [Becton Dickinson]. Immature DCs were CD14− and sorted by FACSort® as a CD83− population. Mature macrophages were generated by culturing an adherent mononuclear cell fraction in a Teflon beaker for 9 days. These APC populations were co-cultured with HLA-A2.1− influenza-infected monocytes. After 12 hr., the APCs were loaded with Na51CrO4, and used as targets for influenza-specific CTLs in a standard chromium release assay (102,119). Specific lysis indicates that the APC had cross-presented antigenic material derived from the apoptotic cell, leading to the formation of specific peptide-MHC class I complexes on its surface. Specific Lysis=[% killing of APC cross-presenting influenza infected monocytes]−[% killing of APC cross-presenting uninfected monocytes]. Background lysis ranged from 0–8%. Controls included influenza infected and uninfected mature DCs, immature DCs and macrophages. The HLA-A2.1− monocytes used as a source of apoptotic material were also tested as targets to demonstrate the absence of lysis when using a mis-matched target. For the control targets, specific Lysis=[% killing of influenza infected APC]−[% killing of uninfected APC]. Background lysis ranged from 0–5%. Maximal influenza specific killing was determined using T2 cells [a TAP−/−, HLA-A2.1+, class II− cell line] pulsed for 1 hr with 1 M of the immunodominant influenza matrix peptide, GILGFVFTL as targets (123). Responses varied from 25–60% as a function of the individual's prior exposure to influenza.
RT-PCR. RNA was purified from highly purified sorted cell populations of immature and mature DCs as described above. Messenger RNA for β3, β5 and CD36 were identified using an one step RT-PCR reaction [Titan kit, Boehringer Mannheim]. The forward primer 5′-TGAGAAGTGCCCCTGCCC was used for both β3 and β5. The reverse primers 5′-GTTGGCTGTGTCCCATTTTGCT and 5′-TTGTAGGATTTGTGAACTTG were used for β3 and β5 to obtain 438 bp and 509 bp products, respectively [primer sequences were generously provided by S. Silletti]. The forward primer 5′-GGGAATTCATATGAAATCATAAAAGCAACAAACAT and the reverse primer 5′CGGAATTCTACATTTCACTTCCTCATTTTCTG for CD36 yielded a product of 392 bp (124). The RT reaction was carried out for 30 minutes at 56° C. followed by 30 cycles of amplification. After 30 cycles of PCR the samples were visualized on an agarose gel.
Immature DCs efficiently phagocytose apoptotic cells. Based on previous observations that immature DCs are the cells responsible for capturing antigen (106), we predicted that apoptotic cells would be engulfed best by immature DCs. To test this hypothesis, we established a phagocytosis assay which allowed us to visually detect the uptake of apoptotic cells, and compare the phagocytic capacity of immature DCs, mature DCs and macrophages. Briefly, immature DCs were prepared by culturing a T-cell depleted fraction from peripheral blood in the presence of IL-4 and GM-CSF. Mature DCs were generated with the addition of monocyte conditioned medium [MCM] and these cells expressed the cell surface DC restricted maturation marker CD83 (113,114,125). Macrophages were prepared by culturing a plastic adherent cell population in Teflon beakers for 3–9 days. As a source of apoptotic cells we used influenza infected monocytes (102); virus infection induces apoptotic death in these cells within 6–10 hours (102,120,121). Monocytes were first dyed red using PKH26-GL [Sigma Biosciences], and then infected with influenza virus as previously described (119). After 6–8 hours, the various APCs were dyed green using the fluorescent cell linker compound, PKH67-GL [Sigma Biosciences], and co-cultured with the apoptotic cells at a ratio of 1:1. After 2 hours at 37+ C., co-cultures of cells were analyzed by FACScan® analysis, allowing for quantification of phagocytic uptake as double positive cells. 80% of the macrophages, 50% of the immature DCs, and less than 10% of the mature DCs engulfed the apoptotic monocytes after 2 hours of coculture [
To confirm that this FACS® assay was measuring phagocytosis, we carried out the assay at 4° C. and in the presence of inhibitors of phagocytosis. Both low temperature [
Only immature DCs cross-present antigen from the apoptotic cell on class I MHC. We next correlated the phagocytic capability of macrophages and DCs with their ability to cross-present antigenic material derived from apoptotic cells. The cells were prepared from HLA-A2.1+ donors (113,114), co-cultured with HLA-A2.1− influenza-infected monocytes for 12 hr and then loaded with Na51CrO4 for use as targets for influenza-specific CTLs (102,119). Specific lysis indicates that the APC cross-presented antigenic material derived from the apoptotic cell, by forming specific peptide-MHC class I complexes on its surface [
While mature DCs were efficient targets when infected with influenza, they were unable to cross-present antigens, presumably because they had down regulated the ability to phagocytose the apoptotic monocytes. The immature DCs, however, did cross-present antigens from apoptotic cells. Furthermore, if the immature DCs were co-cultured with the apoptotic cells in the presence of MCM, a maturation stimulus, they were even better targets. This is possibly due to the up regulation of costimulator and adhesion molecules 108,126), or the increased stability of peptide-MHC I complexes. Given that maximal uptake of apoptotic cells by immature DCs occurs between 2–4 hours [
Notably, macrophages which efficiently phagocytose apoptotic cells [
Immature DCs can be distinguished from macrophages by intracellular expression of CD83 and a unique profile of phagocytic receptors. We investigated the possibility that immature DCs might phagocytose apoptotic cells via pathways distinct from macrophages. To clearly distinguish these cells we characterized them phenotypically. Immature DCs are distinguished by the absence of both CD14, a macrophage restricted marker, and CD83, a maturation marker for DCs (125). We have extended the use of CD83, finding that immature DCs can be distinguished from both macrophages and mature DCs by their intracellular expression of CD83 [
These APC populations were examined for receptors involved in phagocytosing apoptotic material [Table 1]. These include: αvβ3 and CD36 which act as co-receptors, for engulfment of apoptotic neutrophils and lymphocytes by macrophages (127,128); and CD14 which has been implicated in the uptake of apoptotic cells by macrophages (129). While studying the immature DC populations, we identified a discrepancy in the expression of the αv and β3 integrin chains and investigated the possibility that αv was dimerizing with an alternate β chain. Using antibodies which recognize combined epitopes of the αvβ3 and the αvβ5 heterodimers, we noted the selective expression of αvβ5 on immature DCs [
To evaluate whether this down regulation could be observed on the level of mRNA expression, we performed RT-PCR using primers specific for β3, β5 and CD36 [
αvβ5 and CD36 mediate phagocytosis of apoptotic cells in immature DCs. To demonstrate a direct role for αvβ5 in the recognition of apoptotic cells by immature DCs, we performed the phagocytosis FACS® assay in the presence of antibodies specific for αvβ5 [
While some inhibition of phagocytosis was observed when using αvβ3 this may in part be due to transdominance and/or the effect on the pool of free αv (130). For example, anti-αvβ3 antibodies suppress the intracellular signaling of the α5β1 integrin (131). Alternatively, αvβ3 and αvβ5 may be working cooperatively in the immature DCs. We therefore tested combinations of anti-αvβ3 and anti-αvβ5, but did not observe an increase in the inhibition of phagocytosis. The low receptor density of αvβ3 on DCs [average MFI of 7+/−2, Table 1] also makes it unlikely that this integrin heterodimer is involved in the engulfment of apoptotic cells by immature DCs.
Our data do not exclude a role for other receptors in the phagocytosis of apoptotic cells e.g. the putative phosphatidylserine receptor or the lectin receptor (100). In fact, other receptors are probably involved as blocking observed did not exceed 60%, even when combinations of all relevant mAbs were tested [data not shown]. CD14 is unlikely to be involved in the DC's engulfment of apoptotic cells, as DCs do not express this receptor [Table 1]. In macrophages, phagocytosis of apoptotic cells was inhibited by antibodies to αv, β3, αvβ3 and CD36 but not by antibodies to β1, β5 or αvβ5 [data not shown]. This correlates with published data (101,128).
Cross-presentation of antigens to CTLs appears to have two critical features. It is mediated by DCs and apoptotic cells are the preferred source of antigen (102). The requisite stage of DC development for the acquisition of apoptotic cells is the immature phase. In fact, immature DCs, are 4–5 times more efficient than mature DCs in phagocytosis, a feature which also correlates with their ability to cross-present antigen. This exogenous pathway for class I MHC loading is highly effective: relatively few apoptotic cells [ratio of 1 per 10 DCs] are needed to charge the DCs as efficiently as the live replicating virus; exposure of 3–12 hours is sufficient for generating a peptide-MHC complex that is capable of activating CTLs; and it is relatively indiscriminate, as the cellular source can be allogeneic cells or xenogeneic cells (102,132). We believe our earlier studies with mature DCs are explained by the fact that our cell populations were asynchronous and that only by sorting these cells have the differences become apparent. Based on the findings presented here, we suggest that the peripheral tissue DC, exemplified by the immature DC, has an additional important role. It is responsible for phagocytosing cells within tissues which undergo apoptosis [e.g. secondary to viral infection; during normal cell turnover] and migrating to the draining lymph nodes where appropriate T cells are engaged. This pathway may be employed for stimulating or tolerizing CTLs and can account for the in vivo observations of cross-priming of tumor and viral antigens (104,133) and cross-tolerance of self proteins (105,134) in their requirement for a bone marrow derived APC.
A sharp distinction was also demonstrated between immature DCs and macrophages in the handling of apoptotic material. While macrophages are more efficient at phagocytosing apoptotic cells than immature DCs, they fail to induce virus-specific CTLs (102). In fact, they cannot even generate effective levels of peptide/MHC I complexes. In a short-term assay, influenza-specific CTLs could not kill macrophages co-cultured with apoptotic cells. Therefore, macrophages degrade rather than cross-present the ingested apoptotic cells.
Additional evidence exists that macrophages process apoptotic cells differently from DCs, and prevent an immune response. Two groups have demonstrated that phagocytosis of apoptotic cells suppresses a subsequent inflammatory response to LPS stimulation. The macrophage's cytokine profile is skewed toward the synthesis of IL-10, IL-13 and TGF-β, while the production of proinflammatory cytokines such as TNF-α, IL-1β, and IL-12 is down-modulated (136,137). Therefore, the resolution of inflammation is dependant on at least two pathways for removal of apoptotic cells: via macrophages which subvert and suppress proinflammatory responses; and via DCs, which stimulate T cell responses that clear pathogens responsible for the induction of the apoptotic death.
The αvβ5 integrin receptor may be pivotal in the distinctive handling of apoptotic cells by immature DCs versus. macrophages in that it's expression is restricted to the former. We suggest that the unique profile of receptors expressed by immature DCs affects trafficking of phagocytosed apoptotic cells, and consequently facilitates cross-presentation. We have previously shown that NH4Cl inhibits the DCs ability to process antigen derived from apoptotic cells, suggesting that processing in an acidic vesicle [e.g. CIIVs or MIICs] is required (102,132). Indeed, class I MHC may interact with processed antigens in such a compartment, as MHC I molecules have been described in association with invariant chain (138), and can recycle from the cell surface to class II vesicles (139). Additionally, there may be as yet undescribed routes whereby antigens within vesicles can enter the classical endogenous pathway as described recently for antigens derived from the ER (140).
αvβ5 and αvβ3 have both been described to be important in angiogenesis, cell adhesion, migration and now in their ability to phagocytose apoptotic cells. αvβ3 is critical in the phagocytosis of apoptotic cells in macrophages, where it acts in a cooperative way with CD36 and thrombospondin [TSP], collectively forming a ‘molecular bridge’ (142). Recently, it was reported that αvβ5 but not αvβ3 is critical for the engulfment of rod outer segments [ROS] by CD36+ retinal pigment epithelial cells (143,144). This phagocytic system is also inhibited by anti-CD36 antibodies, suggesting that αvβ5, like αvβ3, might cooperate with CD36. Taken together with our observations, TSP, or possibly other soluble factors, may serve to bridge CD36, αvβ5 and the apoptotic cell.
Although similarities in function exist, αvβ5 can be distinguished from αvβ3 in its use of various ligands [e.g. VEGF vs. bFGF]; by the requirements for activation; and by the intracellular signaling pathways [e.g. indirect activation of PKC] (50). Also significant is the fact that the cytoplasmic domains of the two β chains are the portions which show the most considerable diversity (51). Thus, it is possible that the distinct use of the αvβ5 vs. αvβ3 integrin receptors might account for the specialized functions of DCs in the route by which apoptotic material is trafficked and presented. In other words, differential expression of αvβ5 may be responsible for the DCs ability to cross-present antigenic material derived from apoptotic cells, whereas macrophages scavenge and degrade such material.
We have shown in the previous Example that engulfment of apoptotic cells by immature DCs is a receptor mediated phagocytic event, as it is inhibited by EDTA, Cytochalasin D and low temperature (see Example 7)(147). Immature DCs express little αvβ3, instead they employ an alternate αv heterodimer, the αvβ5 integrin receptor, for the efficient internalization of apoptotic cells. As is the case in the macrophage receptor complex, αvβ3/CD36, we believe that the αvβ5 integrin receptor works in concert with CD36. The expression of this receptor pair is restricted to the immature DC and upon maturation, CD36 and αvβ5 protein expression and their mRNA expression are both down regulated. This expression pattern fits what is known about DC biology—the immature cell is involved in antigen capture, while the mature stage is characterized by poor antigen acquisition, but instead is efficient at activating T cells.
In Example 7, we demonstrated that the uptake of apoptotic human monocytes, HeLa cells, and a murine macrophage cells line (RAW) by immature DCs are all inhibited by monoclonal antibodies specific for αvβ5, αv, β5 and CD36. While we do not have direct evidence for the αvβ5/CD36 interaction, we know that addition of blocking antibodies for both receptors to DCs/apoptotic cells co-cultures does not result in increased inhibition. This suggested to us that these two receptors might be working in concert. Maximal inhibition achieved in the in vitro phagocytosis assay was never greater that 60%, suggesting that additional receptors are involved. Candidates include, the complement receptors CR3 and CR4, scavenger receptor A, a putative lectin receptor (possibly DEC-205), and a still undefined phosphatidylserine receptor (Reviewed in (149, 150)).
To better define the αvβ5/CD36 receptor complex on immature DCs, we have begun to search for a DC-restricted bridging molecule as it is unlikely that TSP-1 is the soluble factor involved in the αvβ5/CD36 receptor complex. Notably, neither the addition of antibodies specific for TSP-1, nor the addition of purified TSP to DC/apoptotic cell co-cultures has an inhibitory effect (data not shown). In contrast, both are known to inhibit engagement with CD36 on macrophages, thus blocking the phagocytosis of apoptotic cells. Additionally, we have begun to investigate the role for IAP-1 in the activation of the αvβ5 integrin.
Based on the data described herein regarding the characteristics of a specific molecular bridge between apoptotic cells and DCs we believe that Lactadherin, a major glycoprotein of the human milk fat globule membrane, (151–153) has several properties which suggest it may be a component of such a molecular bridge. Lactadherin has many of the properties required for acting as a molecular bridge between the immature DC and the apoptotic cell as milk fat globules (MFGs) and apoptotic cells share much in common. The membrane surrounding the MFG has the same orientation as the membrane of an apoptotic body/bleb (the outer leaflet of both is topologically identical to the plasma membrane); the external leaflet of the membrane encapsulating the droplet contains PS (likely due to a lack of flippase activity inherent to the MFG); and oligosaccharides comprised of fucose, galactose, N-acetylglucosamine and N-acetylgalactosamine (many of the same carbohydrate motifs found on the surface of dying cells) (152–154). The milk fat droplets are secreted from the lactating epithelial cells by a process of encapsulation in plasma membrane and is subsequently engulfed by the gut epithelium of the nursing child (155). Indeed, the characteristics which make Lactadherin successful at bridging the MFG with the gut epithelium are similar to those which are likely facilitating uptake of the apoptotic cells by the DC.
Supporting data: A role for IAP-1.
Another proposed factor involved in the αvβ5/CD36/Lactadherin complex is integrin associated protein-1 (IAP-1). IAP-1 is involved in the activation of the αvβ3/CD36/TSP-1 complex on macrophages. IAP-1 is expressed on virtually all cells; it is present on immature DCs (data not shown); and likely interacts with other αv integrins as IAP-1 and β5 have been co-immunoprecipitated (195). To test the hypothesis that IAP-1 is responsible for the activation of the αvβ5 /CD36 complex on immature DCs, blocking studies with monoclonal antibodies and pertussis toxin (PT) were performed—we tested the effect of these factors on the phagocytosis of apoptotic cells by immature DCs (see Example 7 for experimental details of phagocytosis assay).
Antibodies specific for IAP-1 (clone B6H12, Chemicon International Inc.) inhibited the phagocytosis of apoptotic cells, however the blocking observed was significantly less than that observed when using anti-ad antibodies (TABLE II). PT, the major virulence factor of Bordetella pertussis inhibits heterotrimeric Gi-proteins, also blocked the phagocytosis of apoptotic cells by immature DCs (TABLE II).
Taken together, our data suggests that αvβ5/CD36/IAP-1/Lactadherin receptor complex on immature DCs may be responsible for the efficient engulfment of apoptotic cells. Similar, but as yet unidentified factors could also serve to enhance the uptake of apoptotic cells (or fragments thereof) in our antigen delivery system.
Preparation of DCs. DCs were prepared as previously described (171–173). In brief, DCs were generated from T cell depleted PBMCs by culturing cells for 5–6 days in the presence of 1,000 U/ml granulocyte and macrophage colony-stimulating factor [GM-CSF, Immunex] and 1,000 U/ml IL-4 [Schering-Plough Corp. Union, N.J.]. RPMI 1640 supplemented with 20 μg/ml gentamicin, 10 mM HEPES, [GIBCO] and 1% autologous plasma [heparinized] was used for cell culture. Cultures were supplemented with cytokines on days 2 and 4. On day 5–6 nonadherent immature DCs were collected and transferred to new six well plates. Mature DCs were generated by the addition of 50% v/v monocyte conditioned medium [MCM] on the day of transfer and harvested on days 8–9.
Induction of Apoptosis and Necrosis. 293 cells were dyed red with PKH 26 according to the manufacturer's protocol [Sigma Biosciences, St. Louis, USA]. UV-triggered apoptosis was induced using a 60UVB lamp [Derma Control Inc]. Cells were incubated for 6 hours following irradiation to allow the cells to undergo apoptosis. Necrosis was achieved via repeated freezing and thawing. Immature DCs were dyed green with PKH 67 and then co-cultured with the apoptotic and necrotic cells for 3 hours at 4° or 37C°. Phagocytosis of apoptotic and necrotic cells by immature DCs was defined by the percentage of double positive cells by FACS-Analysis as previously described (176). UV-triggered apoptosis was also induced using a 60 mJ UVB lamp [Derma Control Inc.], calibrated to provide 2 mJ/cm2/sec. Necrosis was achieved via repeated freeze [−80C° C.] thawing [+37° C.]. Necrotic cells were trypan blue positive and demonstrated a distorted or fragmented morphology.
Cell lines. The following cell lines were used: Human cell lines consisted of EBV transformed B-lymphocyte cell lines [B-LCL cells], melanoma cells [SK29 cells], and kidney adenocarcinoma cells [293 cells] and were cultured in RPMI supplemented with 10% FCS. Mouse cell lines [B16 melanoma cells, L-cells, RAW cells, 3T3 cells] were grown in DMEM supplemented with 10% FCS.
Monoclonal antibodies. Monoclonal antibodies [mAbs] to the following antigens were used. CD83 [Immunotech, Coulter, Marseille, France] and DC-LAMP [generously provided by Dr. S. Lebecque, Schering Plough, Dardilly, France] are markers expressed primarily by mature DCs. CD86, HLA-DR, CD40, CD25, CD8, CD14 were obtained from Becton-Dickinson [Mountainview, Calif.]. Isotype control mAbs included IgG1, IgG2a and IgG2b [Immunotech, Coulter, Marseille, France]. The secondary antibody was PE-conjugated Fab′2 goat anti-mouse IgG heavy and light chain [Jackson, ImmunoResearch, Laboratories, Inc., Baltimore, USA]. The DC cell populations were phenotyped with the panel of mAbs listed above and analyzed on a FACScan [Becton Dickinson, Mountain View, Calif.]. For intracellular staining cells were first fixed in 1% paraformaldehyde and then permeabilized with 0.5% saponin before incubation with the primary antibody.
Co-culture of apoptotic or necrotic cells with immature DCs. Apoptotic or necrotic cell lines were added at various ratios to day 5 or 6 immature DCs. After 48 hours of coculture the DCs were assayed for their T cell stimulatory capacity. For the superantigen dependent assay DCs were irradiated with 3000 rad prior to addition to syngeneic T cells in the presence of 0.1 ng/ml SEA. After 3 days, 4 μci/ml 3H-thymidine was added for 16 hours. We observed background proliferation in 5 day co-cultures, of immature DCs and allogeneic T cells in the MLR, probably because of some DC maturation as a result of DC-T cell interactions. To avoid this, we fixed immature DCs after exposure to dying cells with 1% paraformaldehyde for 30 min on ice, washed extensively and added them in graded doses to allogeneic T cells. After 4 days, 4 uci/ml 3H-thymidine was added for 16 hours. Immature and MCM matured DCs served as controls.
The immune system has to contend with two types of cell death and their consequences. Necrosis, characterized by cell fragmentation resulting from severe and sudden injury, leads to the release of toxic intracellular contents which may induce inflammation (156). Apoptosis, an energy dependent and synchronized event, is considered non-inflammatory due to rapid scavenging by macrophages (156–158). Necrotic and apoptotic cells are also phagocytosed by dendritic cells, potent initiators of immunity, which induce T cell responses to antigens derived from these dying cells (159–161). Uptake is restricted to the immature stage of development when DCs are well equipped to acquire antigen but express low levels of the requisite MHC and costimulatory molecules needed for T cell stimulation (159). Upon receipt of a maturation signal, DCs downregulate antigen acquisition, express higher levels of costimulatory and MHC molecules and become stably differentiated to activate resting T cells. Maturation can be triggered by multiple stimuli including LPS, contact allergens, bacteria and viruses, cell products [monocyte conditioned medium-MCM, TNFα, IL-1β, PGE2, IFN-alpha (162–168)] and signalling molecules [CD 40L (169–170)]. Since it is the immature DC that captures antigen most efficiently, we investigated whether the uptake of dead or dying cells could initiate immunity by inducing DC maturation.
To generate immature DCs, we cultured freshly isolated blood monocytes in GM-CSF and IL-4 for 6 days. These cells are characterized by low levels of HLA and co-stimulatory molecules [CD40, CD86 (159)] and the DC restricted, maturation associated markers CD83 and DC-LAMP [lysosomal associated membrane glycoprotein] (174). We first verified that immature DCs phagocytosed necrotic cells comparably to apoptotic cells (175). A FACS assay was employed where green PKH26-GL labeled DCs are visually assessed for their ability to take up red PKH67-GL labeled apoptotic or necrotic tumor cells [kidney adenocarcinoma, 293 cells] at a ratio of 1:1 (176). Over 40% of immature DCs phagocytosed either apoptotic or necrotic cells within 3 hours [
We next monitored the effects of dying cells on the maturation of DCs. A panel of human cell lines was tested including a melanoma cell line [SK29 cells], kidney adenocarcinoma cells [293 cells] and EBV transformed B-lymphocyte cell lines [B-LCL] [
To define the nature of factors in necrotic cells that were responsible for inducing maturation, we collected supernatants from necrotic and apoptotic cell lines, filtered them through a 0.45 micron filter and added these to immature DC cultures. Supernatants from necrotic but not apoptotic cells induced maturation [
Mature DCs are potent stimulators of T cells, the most straightforward assays being the induction of allogeneic T cell proliferation in the mixed leukocyte reaction [MLR] and superantigen dependent T cell proliferation (179, 180). After a 48 hour exposure to dying cells or their respective supernatants, DCs were washed, and added in graded doses to 200,000 allogeneic or syngeneic T cells, in the latter case together with 0.1 ng/ml staphylococcus enterotoxin A [SEA]. Strong stimulation of allogeneic and superantigen stimulated syngeneic T cells was observed with DCs that had been matured with necrotic cells, their respective supernatants or with MCM [
While a role for macrophages in the phagocytic clearance of both apoptotic and necrotic cells is well known, there is little information on how DCs respond to dying cells. We find striking differences in the handling of dying, transformed cell lines depending on the mechanism of cell death. Necrotic cells selectively induce maturation of DCs. We would predict that in vivo, uptake of necrotic tumor cells would lead to the initiation of T cell responses to antigens processed by the DCs. These responses are likely to be CD4+ rather than CD8+, as antigens derived from necrotic cells do not induce CTLs, at least in vitro (160). Phagocytosis of apoptotic cells by DCs failed to induce maturation, the consequences of which may be the induction of tolerance to self or tumor antigens (184–186). Phagocytosis of apoptotic cells, however, may lead to T cell immunity if followed by a maturation signal. We have shown that DCs phagocytose apoptotic cells and present antigens [e.g. viral and tumor antigens] from these sources to both CD4+ and CD8+ T cells. See, Examples 1–5. Given that phagocytosis of apoptotic cells does not mature DCs, signals provided by necrotic cells in the enviroment, such as cytokines [e.g. TNF-α, IL-1β, IFN-α released by virus infected cells], inflammatory products [e.g. LPS, bacterial cell walls] and CD4+ T cells [such as CD40-L/CD40 interactions (169, 170)] would be required to mature the DCs, thus allowing for the full activation of T cells. In fact in in vivo animal models of cross-priming and cross-tolerance, induction of CTLs requires CD4+ help [(184, 187–189) and data in vitro not shown].
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